The objective of this proposal is to learn what mechanisms of cell death operate during in vivo development of the nervous system, as only the broadest outlines of the mechanism(s) of cell death are known in higher vertebrates. Naturally occurring cell death, as it occurs during development of the nervous system, has different morphological characteristics, nuclear or cytoplasmic, as we have previously described in avian ciliary ganglion (CG) neurons . We will investigate whether the different types of cell death are mediated by similar or different mechanisms, and will characterize the two morphological types of death during CG development in vitro (tentatively referred to as "apoptosis" and "necrosis"). We have developed a tissue culture system that mimics the conditions of target competition in vivo, and will compare the mechanisms operating in this system with those in neurons deprived o trophic support (CIPE, CNTF, and GPA) in vitro, and with cell death in the chick embryo. Perturbations of these mechanisms in vivo and in vitro will allow us to define and clarify the conflicting hypotheses that have been proposed regarding the mechanisms of cell death (apoptosis vs. necrosis). A novel aspect of this proposal is that we will study the cell death process using morphological, electrophysiological and molecular techniques. Furthermore, these techniques will be used in the same cell(s) at critical times (ie. measurements of intracellular Ca++will be made with Fura-2, followed by electrophysiological measurement of Ca++ currents and completed by histological observations of DNA fragmentation). We will characterize DNA degradation using agarose electrophoresis, and the TUNEL and COMET techniques, correlating these and ultrastructural changes with some of the functional mechanisms operating during cell death, such as ionic membrane permeability changes (Na+ and Ca++) and changes in intracellular Ca++ levels. We will then begin to characterize some of the genes that might be expressed, such as bcl-2, and which are perhaps required for survival in vertebrates. These questions are central to determining whether the neuronal degenerative processes occurring in such diseases as Alzheimer's and parkinson's is due to apoptosis (programmed death) or necrosis (death due to injury or toxicity). This information may be ued to develop new treatments or drugs to ameliorate the symptoms of these diseases which affect a large number of patients.